Identification of tumor antigens has paved the way for detection of tumor antigen-specific T cells in cancer patients. However, the developments of therapeutic cancer vaccines are only with limited effects. Among the many factors, tumor- and vaccine-induced expansion of regulatory T cells has been considered to be one major obstacle. Depletion of Treg has led to increased tumor immunity in mice. Anti-CTLA-4 antibody-treatment has shown synergy with cancer vaccine. In anti-CTLA-4 treated cancer patients, Treg-depletion in tumor lesion correlates with therapeutic response. However, progress is tempered by severe autoimmune side effects associated with anti-CTLA-4 antibody therapy. Thus, developing novel strategies to enhance cancer vaccine efficacy is essential for vaccine-based cancer immunotherapy. IL-27 is an anti-inflammatory cytokine that down-regulates autoimmune Th17 response and induces IL-10 expression in T cells. However, accumulating evidences from animal studies have indicated that both endogenous and exogenous IL-27 inhibit tumor growth. Recently, we have obtained compelling evidence that IL-27 may be used as a novel therapeutic for cancer. First, we have found that IL-27 induces stem cell-like Th1/Tc1 effectors with better survival and anti-tumor activity. Second, in mice treated with recombinant adeno-associated virus (rAAV)-delivered IL-27 (AAV-IL-27), we have found a dramatic reduction of regulatory T cells and significant inhibition of tumor growth. Based on these strong data, we hypothesize that IL-27 in combination with cancer vaccine can lead to optimal amplification of tumor-reactive T cells, and IL-27-based vaccination has the potential to be used for cancer therapy without causing autoimmunity. To test this hypothesis, we will first evaluate the mechanisms of AAV-delivered IL-27 in Treg-depletion and tumor growth inhibition. We will then determine if AAV-expressed IL-27 have synergistic effects with cancer vaccines in inducing anti-tumor immunity, and if AAV-IL-27 can be replaced with recombinant IL-27-Fc fusion proteins in combination therapy. The autoimmune side effects in combination therapy will be evaluated using autoantibodies, inflammation and tissue destruction as the major readouts. The proposed studies represent a new approach combining the unique abilities of IL-27 with cancer vaccines to induce tumor regression.